Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

Abstract

Structural changes occur in the ␣␤-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a ␤-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with ␣-tubulin from stacking onto a ␤-tubulin ␤ sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the ␤-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.cytoskeleton ͉ inhibitors ͉ microtubules

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

“…The possibility that the structure of tubulin around the colchicine binding site would change and the ligands binding affinity would remain the same due to compensations of perturbation effects appears unlikely. This strongly supports the proposal (27) and other evidence (29,31) suggesting that unassembled GTP-tubulin is curved, instead of as straight as in microtubular sheets (22) and that its structure is close to that of tubulin in T 2 RB3 (20,21). Our results do not exclude possible GTP-induced structural changes at another zone of the tubulin dimer.…”

“…Since the structure of tubulin in liganded and unliganded T 2 RB3 complexes is curved (this work and Ref. 21) the simplest interpretation is that the liganded and unliganded tubulin dimers are similarly curved. The possibility that the structure of tubulin around the colchicine binding site would change and the ligands binding affinity would remain the same due to compensations of perturbation effects appears unlikely.…”

“…In the straight structure, the intermediate domain ␤-sheet (strands S8 to S10) comes closer to the H8 helix. Together with the H7 translation, this leads to a shrinking of the colchicine domain (21). When a ligand is bound to it, the straight conformation cannot be accommodated because of steric clashes the ligand makes with surrounding secondary structure elements (S8, S9, H7, and H8).…”

“…1A) were superimposable to the structures of the T 2 RB3-colchicine complex (20) and to unliganded T 2 RB3 (except for a loop of ␤-tubulin that lies in the colchicine site in the absence of colchicine domain ligands (21)). This shows that binding of these ligands does not modify the conformation of tubulin in the T 2 RB3 complex (21), which accommodates them without subunit or domain displacements in the crystal.…”

“…The synthetic microtubule inhibitor TN-16 also competes with colchicine for binding to tubulin (46). The recent determination of the structure of T 2 RB3 in complex with TN-16 has shown that this ligand overlaps the trimethoxyphenyl ring A of colchicine and is more deeply buried in the ␤-tubulin subunit, thus extending the colchicine binding domain (21).…”